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- EMDB-2030: Repair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguishe... -

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Basic information

Entry
Database: EMDB / ID: EMD-2030
TitleRepair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability
Map datasurface rendered top-view of human 9-1-1 complex
Sample
  • Sample: human 911 complex
  • Protein or peptide: Rad9
  • Protein or peptide: Rad1
  • Protein or peptide: Hus1
Keywordsflap endonuclease 1 / processivity clamp / DNA replication and repair / checkpoint clamp / electron microscopy
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsQuerol-Audi J / Yan C / Xu X / Tsutakawa SE / Tsai M / Tainer JA / Cooper PK / Nogales E / Ivanov I
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability.
Authors: Jordi Querol-Audí / Chunli Yan / Xiaojun Xu / Susan E Tsutakawa / Miaw-Sheue Tsai / John A Tainer / Priscilla K Cooper / Eva Nogales / Ivaylo Ivanov /
Abstract: Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins ...Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.
History
DepositionJan 11, 2012-
Header (metadata) releaseJan 27, 2012-
Map releaseSep 26, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-2030.png

Downloads & links

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Map

FileDownload / File: emd_2030.map.gz / Format: CCP4 / Size: 2.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsurface rendered top-view of human 9-1-1 complex
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour LevelBy AUTHOR: 0.17 / Movie #1: 0.17
Minimum - Maximum-0.15060534 - 0.45708376
Average (Standard dev.)-0.01399173 (±0.03936361)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-42-42-42
Dimensions848484
Spacing848484
CellA=B=C: 213.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z848484
origin x/y/z0.0000.0000.000
length x/y/z213.360213.360213.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-42-42-42
NC/NR/NS848484
D min/max/mean-0.1510.457-0.014

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Supplemental data

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Sample components

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Entire : human 911 complex

EntireName: human 911 complex
Components
  • Sample: human 911 complex
  • Protein or peptide: Rad9
  • Protein or peptide: Rad1
  • Protein or peptide: Hus1

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Supramolecule #1000: human 911 complex

SupramoleculeName: human 911 complex / type: sample / ID: 1000 / Oligomeric state: heterotrimer / Number unique components: 3
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa

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Macromolecule #1: Rad9

MacromoleculeName: Rad9 / type: protein_or_peptide / ID: 1 / Name.synonym: h9-1-1 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 43 KDa / Theoretical: 43 KDa

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Macromolecule #2: Rad1

MacromoleculeName: Rad1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 31 KDa / Theoretical: 31 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: Hus1

MacromoleculeName: Hus1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 32 KDa / Theoretical: 32 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8 / Details: 20mM Hepes, 150mM NaCl, 3% trehalose
StainingType: NEGATIVE
Details: sample adsorbed on continuous carbon and stained with 2% w/v PTA
GridDetails: 200 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 50000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 6.3 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder: eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 297 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 2.54 µm / Number real images: 15

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Image processing

CTF correctionDetails: whole image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 5000
DetailsThe particles were manually selected using boxer

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Atomic model buiding 1

Initial modelPDB ID:

3a1j

SoftwareName: chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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